Use of ozone to aid detection in thin layer chromatography



United States Patent ABSTRACT OF THE DISCLOSURE A method of detecting a colorless organic compound on a developed thin layer chromatographic plate which comprises oxidizing said compound with ozone to produce an oxygen-containing functional group capable of reacting with a suitable reagent to produce color.

This invention relates to a novel process of detecting and determining a variety of organic compounds on a thin layer chromatogram.

Thin layer chromatography is a fairly new development which has proven useful in the separation and determination of the number of components and their identity in an unknown mixture, since known compounds may be run simultaneously on the same chromatogram for comparison. However, its effective application has been somewhat limited to mixtures of compounds carrying functional groups of adequate reactivity for easy detection when treated with an appropriate reagent. Relatively inert compounds, such as parafiins, non-fluorescent aromatic hydrocarbons, aliphatic and aromatic amides and aromatic esters may be detected only with extremely harsh reagents and then with poor sensitivity. Considerable time and effort may be saved if the total number of components can be located and determined at one time While still maintaining adequate sensitivity.

Therefore it is an object of this invention to provicle a process whereby diiferent classes of organic com pounds may be separated and detected on a single chromatogram. I

It is another object to provide such a process whereby colorless, relatively inert organic compounds may be detected and determined on a chr-omatogram.

Still another object is to provide a process whereby a mixture of amines and amides may be detected on a single chromatogram.

The following detailed description is illustrative of a general procedure which may be used for preparing thin layer chromatographic plates although many modifications of equipment and techniques are possible.

An adsorbent, such as silica gel or alumina, is mixed with Water to form a slurry, which is placed on a glass plate and spread evenly to form a thin layer, usually about .25 mm. thick. This may be done by various means. We used an instrument which is available commercially, the Desaga applicator and mounting board. The plate is placed in an oven at 110 C. for an hour, and may be stored in a dessicator over P 0 The plate is marked with a starting line and finish line, conveniently cm. apart using a standard template. The sample or mixture to be analyzed may be placed on the starting line as a dot with a micro pipette, preferably in sufficient concentration to give from .5 to 50 micrograms of material to the plate.

evelopment of the plate to which the sample to be analyzed has been added is achieved by dipping the edge of the plate below the starting line into a dish containing a suitable solvent or solvent mixture, being careful not to immerse the plate deep enough to cover 3,388,974 Patented June 18, 1968 the starting line. The best solvent to use must be determined pragmatically for each mixture. The plate remains immersed in the solvent until the solvent travels to the finish line, whereupon it is removed and dried.

The solvent separates the various compounds in a mixture according to a certain rate. This is determined as the ratio of the distance travelled by the compound from the point of application to the distance travelled by the solvent, i.e. the distance between the starting line and the finish line on the plate. This is called the R) value, and is a constant for each compound in a specific solvent mixture.

The Rf values for colored compounds may be determined directly without further treatment of the developed plate. In the case of colorless compounds, the developed plate must be coated with a spotting agent which colors such compounds to permit determination of the R values thereof. The most commonly used spotting agents are 50% H 50 chlorine p-toluidine and iodine. The 50% H is a very corrosive spray, inconvenient to apply, and also has the disadvantage that it gives non-uniform droplet size which is capable of damaging the thin layer of adsorbent. Furthermore, it is not very sensitive. Chlorine p-toluidine is not sensitive for many compounds, and did not work at all for amines or amides. Iodine is a sensitive spray but fades rapidly.

In accordance with the novel process of the present invention the unknown colorless compounds on a developed thin layer chromatography plate are first oxidized by contacting the plate with ozone. The plate is then coated by spray containing a reagent capable of reacting with compounds carrying oxygen functionality, such as hydroperoxides, peroxides, aldehydes, ketones, or mixtudes thereof to produce a color in the same. Several sprays containing such reagents are known and can be used in combination with the ozone treatment such as, for example, sprays containing 2,4-dinitrophenyl hydrazine, phosphomolybdic acid, N,N-dimethyl-p-phenylenediammoniumdichloride and potassium iodide. We found that sprays containing 2,4-dinitrophenyl hydrazine and phosphomolybdic acid gave excellent sensitivity, but others may be used.

In addition to providing a means for detecting colorless organic compounds including many which are inert, the above process has an additional advantage in that by ozone treatment and proper choice of reagent spray, spots of increased color stability can be obtained, thereby allowing quantitative determination to be carried out.

In accordance with a preferred method of carrying out the present invention, the developed chromatographic plates are exposed to 24% ozone in oxygen for an adequate period, preferably about 30 minutes, by placing them in a tank provided with an ozone inlet tube in the top, and an exit tube leading to an exhaust hood. For unreactive or inert compounds such as paraflinic hydrocarbons or aliphatic amides, the plates are exposed to ozone at an elevated temperature, preferably from 55- C. Any convenient heating system may be employed; we used an oil bath fitted with a thermostat. After the ozone exposure the plate is placed under a gentle stream of inert gas such as air to drive off the excess ozone. This is necessary to prevent or reduce any reaction of adsorbed ozone with the spotting spray. The spotting agent is sprayed on, and the R values determined for each compound.

A large variety of chemical classes were tested by the above procedure and their approximate sensitivities determined. Aromatic compounds have been found very sensitive to the ozone test, which may find applications in the field of air and water pollution.

Sensitivities vary with the structure of the compound to be detected and also within a class of compounds.

3 The best sensitivities were found with aromatics and amines, and in decreasing order, olefins, alcohols, polyether alcohols, and saturated hydrocarbons. Within the class of aromatics, polycyclics are more sensitive than alkyl benzenes or halogenated aromatics.

We also adopted the above process to determine the extent of reaction and product purity during the synthesis of a series of alkyl and aryl substituted oxazolines. A simple and rapid method was needed to distinguish the starting material, ethanolamine, the intermediate product, B-hydroxyamide, and final product, oxazoline. It was desired to effect several types of separations: (a) separation of hydroxyamide from ethanolamine to determine the purity of the hydroxyamide; (*b) separation of oxazoline from the ethanolamine to determine the purity of the oxazoline; and (c) separation of a ternary mixture of ethanolamine, hydroxyamide and oxazoline, to determine extent of reaction.

The separation of hydroxyamide and ethanolamine was effected by thin layer chromatography using ozone for detection as previously described. The developing solvent used was ethanol-ammonia. The plates were carefully dried after development to evaporate the ethanol, which would also react with ozone and give a background color with the spotting agent, dinitrophenyl hydrazine. The unknown solutions may be compared with previously determined Rf values.

Excellent separation of oxazolines from ethanolamine can also be effected using 1:1 acetone heptane as the solvent, or 80:20 ether-benzene. The latter system gives a shorter developing time, about 25 minutes, but will not separate ethanolamine from hydroxyamide as will the acetone-heptane system. Detection in this case can be either ozone plus 2,4-dinitrophenyl hydrazine or ninhydrin.

The separation of the ternary mixture and determination of all compounds was achieved by combining the ninhydrin test for amides with the newly invented ozone test. We found that ozone lowers the sensitivity of the ninhydrin test, so we developed the chromatogram with ninhydrin first, protectively covering the amide area of the plate, determined the Rf values of the ethanolamine and oxazoline, and then exposed the chromatogram to ozone, and detected the oxidation products of the amide with dinitrophenylhydrazine.

The following examples are given to further illustrate the present invention, but it is to be understood that the invention is not to be limited to the details described therein.

Example 1.Sensitivity of various organic compounds to ozone test Thin layer chromatoghaphic plates were made by coat ing glass plates evenly with a .25 mm. layer of silica gel, dried in an oven at 110 C. for one hour and stored over P A solution of the compound to be tested was placed on the plate so that mm. contained .5 to 50 micrograms of the compound. The plates were exposed to an atmosphere of ozone by placing them in a rectangular tank with an aluminum cover plate, provided with an ozone inlet tube extending to the tank bottom and an exit tube leading to a water bubbler and exhaust hood. When a higher temperature is desired, the tank is immersed in an oil bath fitted with a thermostat. The plates were exposed to the ozone for 30 minutes, removed from the tank and placed in a gentle stream of warm air to remove excess ozone. The plate was sprayed with 2,4dinitrophenyl hydrazine (.4% in 2 N HCl). The sensitivities of various compounds and classes of compounds may be seen from the table below. The sensitivities thus determined are somewhat better than those obtained after actual development but will serve as a very good guide in the choice of concentrations required for practical separations.

Concentration in Mierograms Compound 2.5 5 10 Other Aromatics:

n-Decylbenzene Chlorobenzene Anthracene Naphthalene 2,4-dichlorobenzene Alcohols:

n-Decyl alcohol 2-Methylcyclohcxanol Cyclohexylamiue Pyridine Dodecylamine Olefins:

Styrene Oleic acid Saturated Hydrocarbon Teiradocaue Pentadecane Ozone treated at 55 C: at 20. Deealin Stearic acid Ether-Polyether:

Dipropylene glycol. at 20. 1,2diethoxyethane Ethoxylated Alfol (59.5%

ethylene oxide). Amides:

RCONH-CHZCH2OH at 20. Oxazolines:

f 8i RC\ H2 NOTES:

- Not perceptible. Perceptible. Clear spot.

++ Strong spot. Very strong spot.

Example 2.Separation of ethanolamine from fl-hydroxyethylamide The thin layer chromatographic plates were coated as described in Example 1. The plate was marked with a starting line and a finish line, 10 cm. apart. A solution of the sample was placed on the starting line with a micro pipette. The plate was developed by dipping in a solvent solution of ethanol and ammonia (4:1 by volume) until the solvent front reached the finish line, about 70 minutes. The plate was then dried in air and exposed to ozone for 30 minutes as described in Example 1.

The plate was then removed from the tank, held under a gentle stream of warm air, and sprayed with dinitrophenylhydrazine solution. The spots were compared with previously determined Rf values.

Example 3.--Separation of ternary mixture of ethanolamine, fi-hydroxyamide and oxazoline The chromatographic plates were prepared as in Example 1. The solvent used was 1:1 acetone-heptane. After the solvent front reached the finish line, the plate was dried at C. for 5 minutes, and the area where the hydroxyamide was expected was outlined carefully, removing about 5 mm. of adsorbent above and below this area to prevent capillary action of the ninhydrin reagent. The outlined area was covered with a protective strip of aluminum foil or polyethylene, and the plate was sprayed with ninhydrin reagent (.3 gram in butanol containing 3 ml. CH COOH) and'placed in an oven at 110 C. for half an hour to bring out the maximum color. The spots, corresponding to the ethanolamine and oxazoline were marked and their Rf values determined. The protective strip was removed and the plate placed in the ozonizing tank for 30 minutes as described in Example 1. The excess ozone was driven 011 with a gentle stream of warm air and the plate sprayed with 2,4-dinitrophenyl hydrazine solution (.4% in aqueous 2 N HCl). The spots corresponding to the fi-hydroxyamide were measured and their Rf values determined.

Example 4.Detection of various compounds using phosphomolybdic acid of alcohols.

Compounds separated Solvent Mixture 1. Oxazolines from hydroxyamides 1:1 aeetone-heptane.

2. Hydroxyamides from ethanolamine Do.

0 ll 3. CHa-Q-C-OCH; from ortho 4:1 ether-benzene.

and meta isomers.

o [I 4. o-om-Q-o-o on; from ortho and meta isomers.

t s. orn-Q-o-oom from Do.

hydroxyamides.

6. Position isomers of straight chain alcohols Do.

from C to 015 from each other.

7. Primary and secondary alcohols from ethoxy- 1:4 ether-benzene.

lation products of the above alcohols.

We claim:

1. A method of detecting at least one colorless organic compound on a developed thin layer chromatographic plate which comprises exposing said plate to ozone whereby said organic compound is oxidized and thereafter coating said plate with a reagent capable of reacting with the oxygen functionality thus created to produce color in said organic compound.

2. A method as defined in claim 1 wherein said reagent is selected from the group consisting of 2,4-dinitrophenyl hydrazine and phosphomolybdic acid.

3. A method as defined in claim 1 wherein said plate is exposed to ozone at an elevated temperature.

4. A method as defined in claim 3 wherein said elevated temperature is from to C.

References Cited J. M. Miller et al.: Chromatostrips for Identifying Constitutents of Essential Oils, J. Anal. Chem. 25, 1107 (1953).

MORRIS O. WOLK, Primary Examiner.

S. MARANTZ, Assistant Examiner. 

